Bacteriorhodopsin-based photochromic sensor for detection of chemical and environmental toxins

ABSTRACT

A bacteriorhodopsin based chemical sensing architecture based upon the collective response of bacteriorhodopsin and a number of its mutants; the wild type protein and a selection of genetically-engineered variants was able to respond differentially to a selection of amines. The observable response to the presence of a target chemical was manifested through a modulation of bacteriorhodopsin&#39;s photokinetic properties, which are monitored through pump-probe techniques using a custom prototype flash photolysis system. Differential responsivity exists at two levels; (1) bacteriorhodopsin proteins (wild-type and genetically-engineered variants) respond differentially upon exposure of a target chemical, and (2) the response pattern exhibited by the proteins differs from chemical to chemical. This dichotomy forms the basis for a BR-mediated chemical sensing technology that is highly sensitive and selective and may therefore discriminate between different chemicals.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional ApplicationNo. 60/807,729, filed on Jul. 19, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to chemical sensing architectures and,more specifically, to a bacteriorhodopsin based chemical sensor.

2. Description of the Related Art

The motivating force behind sensor technology of any type is health.Whether it be defined in terms of human, machine, building, system, orthe environment, health maintenance is critical in today's society. Thefactor to be sensed depends on the application; mechanical stress,pressure, temperature, light flux, and chemical or biologicalcontamination are all common targets of sensor architectures. Perhapsthe largest motivation in sensor technology development is found withinthe last two examples, the ability to detect compounds, toxins, ororganisms deleterious to human health. A number of different sensorarchitectures are currently being explored, but the most prominent areefforts to mimic biological detection schemes. In an effort to model themechanisms for biological chemical detection (i.e., smell and taste),researchers are moving away from specific detection interactions(favoring detection of a single chemical species or class of molecules),toward architectures that examine the collective responses of largernumbers of sensors that are characterized by non-specific molecularinteractions. These technologies are typically referred to as electronicnoses.

One approach to sensor design that has been explored very little todate, at least from the perspective of electronic nose technology, isthat of hybrid architectures employing biological molecules as an activeelement of the detection scheme. There are several conceivable reasonsas to why this approach has not been widely explored, such as stabilityand state determination. Complex biological molecules are not generallyknown for their inherent stability, and the ability to interrogate forthe purpose of state determination requires some sort of signaltransduction mechanism. Although many proteins act as signaltransducers, few perform that function outside the confines of abiological organism and the transduction mechanisms that are employedinside the organism are not easily adapted to non-biologicalenvironments, at least not in a way that the signal of interest can beamplified and detected. One protein, Bacteriorhodopsin, or BR, does notsuffer from these disadvantages, but has never been successfully adaptedfor use in a commercially viable sensor.

SUMMARY OF THE INVENTION

It is therefore a principal object and advantage of the presentinvention to provide a sensor for detecting the presence of chemicalsusing bacteriorhodopsin.

It is another object and advantage of the present invention to provide asensor for detecting chemicals that is highly selective.

It is an additional object and advantage of the present invention toprovide a sensor for detecting chemicals that is highly sensitive.

In accordance with the foregoing objects and advantages, the presentinvention provides a sensor for detecting chemicals comprising thedetection of changes in the optical response of bacteriorhodopsin in thepresence of a target chemical or compound. The sensor generallycomprises a monochromatic light source for initiating the photo-cycle, asample cube containing bacteriorhodopsin and a target chemical, a firstlens for directing light from the source onto the cube, a detector foridentifying the frequency of light passing through the sample cube, anda second lens for directing light passing through the sample cube ontothe detector. The presence of, and changes in the concentration ofvarious chemicals, may be detected by measuring the effect of theoptical response on various mutants of bacteriorhodopsin.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIG. 1( a) is a schematic of protein structure, illustrating thechromophore, the protein backbone, and key amino acids involved infunction.

FIG. 1( b) is a schematic of the BR Photocycle, illustrating the mainintermediate states, along with λmax and the branched photocycle.

FIG. 2( a) is a diagram of the bacteriorhodopsin photoelectric effectshowing three components.

FIG. 2( b) is a diagram of the bacteriorhodopsin photoelectric effectshowing the movement of a specific amino acid in response to chromophoreisomerization.

FIG. 3 is a graph of the simulated absorption spectra of theintermediaries in the BR photocycle.

FIG. 4 is a diagram of a bacteriorhodopsin mutant matrix sensoraccording to the present invention.

FIG. 5 is a schematic of the optical light path of a probe beamaccording to the present invention.

FIG. 6 is a schematic of a photodiode detector circuit according to thepresent invention.

FIG. 7 is a schematic of the control electronics for a photo-detectionsystem according to the present invention.

FIG. 8 is a schematic of the mechanical design of a sample detectoraccording to the present invention.

FIG. 9( a) is a graph of sample data sets showing O-state modulation asa function of the concentration of 1,2-diaminopropane.

FIG. 9( b) is a graph of sample data sets showing modulation of theM-state decay constant as a function of a selection of amine compounds.

FIG. 10( a) is a chart of differential sensitivity of BR variants to asingle chemical.

FIG. 10( b) is a chart of differential sensitivity of a pair of BRvariants to two different chemicals.

DETAILED DESCRIPTION

Bacteriorhodopsin, or BR, occurs as an integral membrane protein inHalobacterium salinarum, an archaebacterium common in salt marshes wherethe concentration of sodium chloride is >4M. It is typically isolatedfrom the native organism as patches of purple membrane, consisting ofroughly 75% BR & 25% lipid. Its role in nature is to serve as a light tochemical energy transducer by creating a light-induced proton gradientacross the cell membrane. This pH gradient is harnessed by the cell todo work. The chromophore responsible for absorption of light by theprotein is all-trans retinal, a derivative of Vitamin A, which is boundvia a protonated Schiff base to Lys-216. Light induces an all-trans to13-cis isomerization in the chromophore, followed by a series of thermalprotein intermediates characterized by different absorption spectra,vectoral proton transport, and ultimately the reisomerization of thechromophore with consequent re-generation of the bR resting state.

FIG. 1 illustrates the basic photocycle of bacteriorhodopsin upon theabsorption of light, the all-trans retinal chromophore embedded withinthe protein isomerizes to 13-cis, resulting in the formation of theK-state, the only photochemically generated state in the photocycle. Theremainder of the intermediates are thermal, being driven by the chemicalenergy stored in the protein as a result of the initial photonabsorption. During the remainder of the photocycle, L through O, thechromophore is reisomerized and a proton is translocated across themembrane, thereby resetting the bR resting state, and priming theprotein for absorption of the next photon. At room temperature, thephotocycle is completed in roughly 10 ms. Several of the intermediatesare known to be photochemically active, including the M and O states.Upon absorption of blue light, the M state is driven back to the bRresting state. The O-state is the gateway to the branched photocycle,consisting of the P & Q states. These intermediates have applications inthree-dimensional optical memory storage, and are the first trulypermanent intermediates described in the photocycle. A parallel researcheffort funded by NYS Infotonics is evaluating the potential of thebranched photocycle for volumetric holographic and associative memoryapplications. Aside from the branched photocycle, the bR, M, and Ostates are the most important with respect to applications inbiomolecular electronics, for three primary reasons: they are the mosteasily detected intermediates in the photocycle, they are extremelysensitive to the chemical environment experienced by the protein, andthey are the most spectrally distinct intermediates in the photocycle.Furthermore, the M & O states are longest lived of the intermediates,allowing them to accumulate into easily detectable quantities in thenative photocycle. And finally, both of these states have been thetarget of numerous molecular biology studies, which have produced alarge number of genetically engineered variants. These mutant proteinshave yielded a wealth of understanding of both the M & O states, makingthem the best characterized of the BR photocycle. Many of the geneticanalogs are easily produced and have already been evaluated for deviceapplications.

In addition to the BR's optical response to light, the protein alsoexhibits a well defined photoelectric or photovoltaic response uponabsorption of a photon. The origins of the signal involve both themovement of the positively charged Schiff base upon isomerization of thechromophore, as well as a concomitant shift of charge along the retinalpolyene chain, towards the Schiff base. FIG. 2 illustrates the effect,which can be classified based on time scale. The B1 signal has a risetime of less than 5 ps and is associated with the formation of the Kstate. The B1 component is likely attributable to partial chromophoreisomerization and a coupled motion of Arg-82 toward the primarycounterions, Asps 85 and 212. Attempts to correlate B2 and B3 withphotocycle intermediates is difficult and a source of some disagreementin the literature. A secondary photoelectric effect on a millisecondtime scale is associated with proton translocation in the channel (alight-induced photocurrent). Both of these responses to light have thepotential to be exploited for device applications, including sensors,artificial retinas, and as photosensitive microelectronic devices (e.g.,use of the protein as the gate in a traditional MOS field effecttransistor).

BR possesses a number of appealing characteristics: (1) the protein isinherently rugged and robust, and is resistant to both thermal andphotochemical damage (a result of evolving in a harsh environment); (2)high cyclicity (the average number of times the protein can photocycleprior to degradation) on the order of 106 or better, considerably higherthan most synthetic photochromic materials; (3) the protein isinherently radiation hardened and protected from free radicaldegradation; and (4) the primary photochemical event, i.e., absorptionof a photon and consequent formation of the K state as a result ofretinal isomerization, proceeds with a very high quantum efficiency ofroughly 65%. This means that for every 100 photons absorbed by theprotein, 65 will result in a photochemical transition. In addition, thesemi-crystalline arrangement of the protein as an array of trimers inthe membrane also imparts tremendous stability.

FIG. 3 illustrates the absorption spectra of the various BRintermediates, several of which are important to the application of theprotein as an active element in biomolecular electronics. Among theearliest applications of bacteriorhodopsin were holographic films,utilizing the bR and the M intermediates, as mentioned above. Earlyattempts to utilize this binary pair depended upon the ability to extendthe M-state lifetime through chemical means; the M-state is sensitive toany environment or chemical that decreases proton mobility, includinghigh pH, quaternary amines, glycerin, etc. This represented the firstattempt to exploit bacteriorhodopsin's sensitivity to its chemicalenvironment for device applications. Since then a number ofbacteriorhodopsin analogs have been developed that exhibit modulatedresponses to environmental stimuli, through chemical additives to theprotein environment, chemical modification of the retinal chromophore,and genetic manipulation of the protein itself. Perhaps the mostversatile aspect of bacteriorhodopsin's utility as a photoactive elementdevice applications is the ability to interrogate the protein bymultiple means, by examining modulation of either the optical orphotovoltaic properties as a result of external stimuli.

The high quantum efficiency, large oscillator strength, broad-bandabsorptivity, thermal stability, and fast photovoltaic response of theprotein make it an attractive material as the photoactive element inhigh-speed photodetectors. Such applications include high speedtracking, motion and edge detection as well as high resolution imaging.The significant potential of BR as a light-transducing material wasrecently shown in a hybrid protein-semiconductor monolithicallyintegrated transimpedance photoreceiver. In this device, thephotovoltage generated by the protein biases the gate of an amplifyingfield effect transistor (FET), creating a photocurrent signal that isfurther amplified as a current or voltage signal in subsequenttransistor-based amplifying stages. The optoelectronic integratedcircuit (OEIC), uses the protein as a photodetector, demonstratingperformance characteristics comparable to, or better thanall-semiconductor OEIC photoreceivers.

BR is an extremely sensitive register of its environment, and has beenlong known to display an extreme sensitivity to environmental conditionssuch as pH, relative humidity and ionic strength. The effects of thissensitivity are manifested through modulations in the photocycle and/orthe photovoltaic signal. Furthermore, various chemicals havewell-defined effects on the protein. Among the classes of chemicals thatmodulate bacteriorhodopsin's photokinetic behavior include alcohols(methanol, ethanol, propanol, and butanol) and anesthetics. Azides arealso known to modulate BR's photophysical properties. Sensitivity of theprotein to alcohols is manifested in the form of modulations of thelight-induced photocurrent of the protein (i.e., the proton pumpefficiency), and could be enhanced by chemical modification of thechromophore or genetic manipulation of the protein. Generally speaking,chemical agents have several modes by which they can modulate proteinresponse, including direct interaction with, or binding to, the protein,and/or indirect interaction by absorption into the lipids proximal tothe protein.

The basis for a chemical sensor technology employing bacteriorhodopsinis founded in the protein's sensitivity to its chemical environment. Asensor platform based upon one active sensing element would be extremelylimited, and exceedingly impractical. However, bacteriorhodopsin has adistinct advantage over many conventional materials, in that it can bemodified through several approaches. The most powerful approach isgenetic engineering, whereby the protein can be manipulated at thegenetic level, resulting in variants that will exhibit a range ofdistinct properties. There are methods by which proteins can be modifiedtoward specific goals; for example, increased sensitivity to a specificcompound or class of compounds might be introduced through thegenetic-engineering techniques of random mutagenesis and directedevolution. However, given that superior sensor architectures are beingdeveloped through nonspecific sensing elements, production of highlyselective BR variants would defeat the purpose of an electronic nose.

The present invention is based on the capability of wild typebacteriorhodopsin to respond to changes in its chemical environment.Genetically engineered variants would be expected to also exhibitdifferential sensitivity, whereby each would respond differently to agiven target chemical. Monitoring a group of such BR variant proteinsresults in a numeric matrix, or fingerprint, for each target chemicalexposed to the sensor. Denoted as the “Mutant Matrix,” this collectionof BR variants forms the basis for a protein-based electronic “nose.” Itshould also be recognized by those of skill in the art that proteinssuch as proteorhodopsin would behave in like fashion and thus may beimplemented in the present invention.

At least three numeric fingerprints are possible for each chemicaldetected, based upon interrogating the BR-based sensor elements forphotocycle kinetics (of both M and O states) and the photoelectriceffect; the combination of an adequate matrix size with multiple modesof interrogation should help reduce false positive detection events.

The basic element of the present invention is a sensor platform that canquickly and easily measure the protein's response upon exposure to atarget chemical. A chemical sensor must function by observing thephotocycle of bacteriorhodopsin (BR) and its mutants. The method that ismost commonly used to observe the photocycle is a pump-probe experimentknown as flash photolysis. A dim continuous light (probe) passes throughthe sample. The light is too dim to excite the photocycle in the bR. Theprobe light can be either white light to generate a spectral response,or a monochromatic light to generate a kinetic response. Commercialinstrumentation exists to perform this operation (such as that availablefrom OLIS or Edinburgh Instruments). As this instrumentation is largeand expensive, it not suitable to chemical sensor application.Accordingly, the present invention encompasses a new flash photolysissystem and method.

The key item that must be eliminated from the commercially availableinstruments is the pulsed gas laser that is used as the pump beam sourceas such lasers have delicate optics, are large and heavy, are typicallywater cooled, and are extremely expensive. An alternative is asemiconductor diode laser, which is lightweight, rugged, and isrelatively less expensive. The pulse energy derived from a gas laser ison the order of 10-100 mJ, while the diode laser can only produce apulse on the order of 1-10 μJ. The difference is a factor of 10,000 witha corresponding decrease in signal size of the some amount. As a result,the present invention requires a much more sensitive detector.

In current commercial instruments, either a CCD camera or aphoto-multiplier tube is used as the detector. For the purposes of achemical sensor platform, neither of these systems can be used becauseof size and cost. A sensor based on the photodiode must be developed forthe present invention. Finally, the probe light source, which is mostcommonly a high-power tungsten-halogen lamp, must be replaced with anLED. The present invention thus constitutes a functioning flashphotolysis system that uses an LED as the probe light source and asemiconductor diode laser as a pump light source. In addition,absorbance changes are detected using a photodiode amplifier circuit. Adiagram of the probe beam light path is depicted in FIG. 5.

An ultra-sensitive detector circuit is critical to a practical chemicalsensor based on BR. Because photodiodes are current output devices,standard capacitive coupling will not work. Therefore an active ACcoupling scheme such as that published in the application bulletin byBurr-Brown must be used, see Stitt, M. and W. Meinel, OPT201photodiode-amplifier rejects ambient light, in Burr-Brown ApplicationBulletin, AB-061, 1993, hereby incorporated by reference. The presentinvention adds a sample-and-hold amplifier to increase the measurementtime to accommodate long lifetime mutant samples. By using the ACcoupled detector, changes in the probe beam intensity striking thephotodiode smaller than one part in ten thousand can be measured. Thebasic circuit used is shown in FIG. 6. In particular, FIG. 6 depicts ablock diagram of photodiode detector circuit where the servo amp hasbeen drawn as an inverting amp in the diagram for clarity, however, inpractice this is likely a non-inverting integrator. In the presentinvention, the output is followed by a second stage variable gainamplifier to allow for a greater dynamic range in the response ofvarious mutated strains of the BR protein.

The AC coupling feature of the amplifier may be considered an“auto-zero” mechanism. When the sample and hold amplifier is set tosample, the output is forced to zero volts, and then when the amplifieris switched to hold mode, changes in the light intensity striking thephotodiode can pass though as amplified changes in the output voltage. Astep-by-step instruction of now the amplifier is used in practice isgiven below.

First, the sample and hold is set to sample. The photodiode absorbslight from the probe beam and converts the light to current. The currentfrom the photodiode goes to a high gain amplifier. The amplifier feedsan integrator, which increases its output. The integrator output voltageis converted to current. The current is sent to the diode and suppliesthe current needed to balance out the steady state current. The highgain amplifier is no longer amplifying the steady state signal, and theoutput is zero.

Next, the sample and hold is set to hold. The photo-cycle is initiatedby a pulse from the pump laser. The probe beam undergoes a small timedependent change in intensity. This change is amplified by the high gainamplifier. The amplified signal is measured by the micro-controller atspecified time intervals. When the BR has gone back to the ground state,the sample and hold is set to sample, and the system goes back to 1.

A PC to control operations and process the data. The PC communicates toa micro-controller via an RS-232 serial port. The micro-controllercontrols the operation of the apparatus. Controls are provided for thepump beam, the probe beam, the photo-detector, and temperature of thesample. The micro-controller collects data, including the steady stateprobe beam intensity, the time varying probe beam intensity, and thetemperature. These are sent to the PC via the serial port.

For a pump beam, the present invention is set up to use several possiblesources. These include Lasiris lasers, both the diode lasers (635 nM and685 nm) and a Lasiris Diode Pumped Solid State Laser (532 nm). Theselaser modules are inexpensive and highly reliable. The intensity of thelasers varies from 10 to 20 mW depending on the module. High power LEDsmay also be used for pumping. The LEDs tested are similar to thosecurrently being used in traffic lights. The pulse beam time length canbe varied from 0.1 milliseconds to 20 milliseconds. Typically a pulselength of 1.0 millisecond is used.

The probe beam is derived from a standard high-intensity LED. These LEDscome in a variety of wavelengths ranging from 690 m to 430 nm. TheseLEDs provide a very steady light source and are powered by a constantvoltage source with a resistor in series to maintain a constant currentthrough the LED.

The photo-detector used is an OPT 101. This detector has a built inamplifier with a current to voltage gain of one million. The bandwidthis 10 kHz, allowing resolution of time dependent changes in intensity to0.1 milliseconds. The detector is followed by a programmable gainamplifier. The gain varies from 1 to 100. Two PCBs (Printed CircuitBoards) are used for the electronics. The first has the microcontrollerused to communicate with the PC. The second has the control interfaceelectronics and the photo-detection system. The schematic for the secondboard is shown in FIG. 7.

The mechanical design for the apparatus is shown in FIG. 8, wheremeasurements are in inches. As the primary concern is to provide astable, vibration free system the platform is contains attachment pointsto make is easy to use the apparatus on an optical bench. The sample isheld is a standard spectroscopy cuvette (fluorescence), thereby makingit easy to prepare samples that are standardized. Other preparations ofthe protein, such as dried films with or without various matrices tostabilize the protein, polymers, hydrogels, sol-gels, hybrid materials,or other materials that fix the protein in place in a form that enablesinterrogation of the photocycle are also useful.

Using the present invention, the response of the wild type protein andseveral mutants to a number of target chemicals was evaluated. Severalamines were selected for evaluation of protein response because thisclass of compounds has been previously shown to affect BR photokineticsin a reproducible manner. Protein response was gauged in the presence ofthe target chemical in concentrations ranging from 0 ppm to 1000 ppm.Both the M- and O-states were monitored with respect to their rise anddecay at select wavelengths (470 nm and 650 nm). A typical data set forO-state photokinetics is shown in FIG. 9 a. Decay constants were plottedas a function of amine concentration, as illustrated for the M-state inFIG. 9 b. In addition to wild-type BR, similar experiments wereperformed for a number of BR mutants (E204Q, T205A, and T205A/N202S),all of which behaved differently in the presence of the same selectionof amines. To facilitate comparison of BR variants examined over thewide range of chemical concentrations, sensitivity was defined as thefollowing ratio:

$\underset{\_}{{\%\mspace{14mu}{change}\mspace{14mu}{in}\mspace{14mu}{time}\mspace{14mu}{constant}\mspace{14mu}{or}\mspace{14mu}\%{\mspace{11mu}\;}{change}\mspace{14mu}{in}\mspace{14mu}{peak}\mspace{14mu}{height}}\mspace{14mu}}$%  change   in   chemical  concentration

This definition results in a unitless, quantitative method for relatingthe change in output to the change in input, as well as a method tocompare different proteins across a large chemical range. Furthermore, asignificant increase in the sensitivity range could be realized byconsidering only the largest parameter for each target chemical (i.e.,O-state time constant, O-state peak height, and M-state time constant).Sensitivity was shown to differ between different mutants and WT to thesame chemical. However, in order show that it is possible todifferentiate between different chemicals it was also necessary todemonstrate different sensitivities to different chemicals. This wasdemonstrated by compiling the responses for a single protein to multiplechemicals, as well as the responses for multiple proteins (mutants) to asingle chemical, as seen in FIG. 10.

The present invention may be used to test BR mutants for usefulness inscreening a wide variety of chemicals, such as model compounds forchemical warfare agents, as well as a selection of health andenvironmental toxins. The latter categories include toxic industrialchemicals as identified by the NATO International Task Force 25, which,although not chemical warfare agents, could potentially be utilized asweapons in an area of engagement. Examples include, among others, borontrichloride, phosgene, ammonia, hydrogen cyanide, and a variety ofacids. Many of these chemicals can be easily secured without specialpermit.

The BR photoelectric effect as a gauge of the protein's sensitivity tothe presence of chemicals may also be evaluated using the presentinvention. Data analysis techniques often utilized in thecharacterization of chemical sensor architectures and electronic noseswill be performed to facilitate comparison to other popular sensortechnologies currently reported in the literature. Such statisticaltechniques employ multivariant analytical approaches to data sets withmultiple response components (i.e., a number of different measurementsextracted from one sensor reading), including a number of patternrecognition algorithms (e.g., principle component analysis and neuralnetwork approaches).

1. A sensor for detecting a target substance, comprising: a sensorelement comprising bacteriorhodopsin; a probe light source for producinga probe beam directed at said sensor element; a pump light source forproducing a pump beam directed at said sensor element; and a detectorpositioned to receive said probe beam produced by said probe light aftersaid probe beam passes through said sensor element, wherein saiddetector is programmed to determine the intensity of said probe beam andthe optical state of the bacteriorhodopsin when said bacteriorhodopsinis in contact with said target substance.
 2. The sensor of claim 1,further comprising a lens for directing light from said probe lightsource onto said sensor element.
 3. The sensor of claim 2, furthercomprising a second lens for directing light passing through said sensorelement to said detector.
 4. The sensor of claim 3, wherein said sensorelement further comprises a housing containing said bacteriorhodopsin.5. The sensor of claim 4, wherein said housing additionally containssaid target substance.
 6. The sensor of claim 1, wherein said probelight source is a monochromatic light source.
 7. The sensor of claim 1,wherein said detector is programmed to determine the time-dependentchange in both the intensity of said probe beam and the optical state ofthe bacteriorhodopsin when said bacteriorhodopsin is in contact withsaid target substance.